This invention relates to assays of blood cell function, and particularly to assays of dendritic cell function in whole blood.
Dendritic cells (DCs), first identified a quarter century ago by a characteristic xe2x80x9cdendriticxe2x80x9d morphology observable in peripheral lymphoid tissues, Steinman et al., J. Exp. Med. 137:1142-1162 (1973), are now known to be a morphologically-diverse and widely-distributed cell population. Today, these diverse cells are collectively distinguished by a common function: dendritic cells are the most potent antigen-presenting cells (APCS) of the mammalian immune system, and alone among the various antigen-presenting cells appear capable of triggering a primary T lymphocyte response.
This singular ability to prime a T cell-mediated immune responsexe2x80x94combined with a potent ability to present antigen to activated T cellsxe2x80x94has implicated dendritic cells as potential reagents for immune-based therapies, as well as likely targets for therapeutic intervention in the treatment of various immune-mediated disorders.
For example, WO 97/24438 describes compositions and methods for co-culturing dendritic cells with T lymphocytes and protein antigen in vitro, thus driving the ex vivo antigen-specific activation of T cells. The activated T cells are then administered autologously to effect an antigen-specific immune response in vivo. Similarly, WO 97/29183 describes a method of activating T cells in vitro by contacting the T lymphocytes with DC that directly express an antigenic protein from a recombinant construct. Again, the activated T cells are intended for autologous infusion. Specific application of DC-driven ex vivo T cell activation to the treatment of prostate cancer is described and claimed in U.S. Pat. No. 5,788,963. In yet another approach, Nemazee, U.S. Pat. No. 5,698,679, describes and claims immunoglobulin fusion proteins that deliver antigenic peptides to targeted antigen presenting cells (APCs), including dendritic cells, in vivo.
Dendritic cells have also been implicated as important in the pathogenesis and pathophysiology of AIDS. One type of DC, the Langerhans cells (LC), is generally believed to be the initial cell type infected with HIV following mucosal exposure to virus. DC are believed to act not only during the initial phase of HIV disease, but also during the chronic phase, facilitating infection and depletion of T lymphocytes. Zoeteweij et al., J Biomed Sci 5(4):253-259 (1998). DCs in lymphoid mucosa may represent a key reservoir of viral nucleic acid and virions throughout the course of the disease. Grouard et al., Curr. Opin. Immunol. 9(4):563-567 (1997); Weissman et al., Clin. Microbiol. Rev. 1997 10(2):358-367 (1997). In vitro methods for screening pharmaceutical candidates for agents that abrogate HIV infection of DC are described and claimed in Steinman et al., U.S. Pat. No. 5,627,025.
Yet despite their importance to the normal mammalian immune response and in immunopathology, DCs have been difficult to study, and particularly difficult to study in their native milieu.
The difficulty stems in part from the rarity of dendritic cells. Although widely distributed, DC are sparse, even in lymphoid tissues, and represent no more than about 0.3%-0.5% of nucleated cells in human peripheral blood.
A further difficulty arises from the absence of DC-specific cell surface markers that would readily permit the positive immunoselection of DCs from mixed populations of cells.
Extensive efforts to identify surface markers that define DCs have been only partially successful. As a result, DCs are presently identified by multiple-marker panels, with identification based primarily on the absence of staining with markers for other lineages (i.e., as linxe2x88x92 cells) . The result is that typical DC immunopurification protocols require at least one immunodepletion step, eliminating cells of various nondendritic blood lineagesxe2x80x94lymphocyte, monocyte, granulocyte, and NK lineages, e.g.xe2x80x94coupled with at least one immunoenrichment step. The immunoenrichment step may, for example, include selection for CD4+ cells (Blood Dendritic Cell Isolation Kit, Miltenyi Biotec #468-01, Auburn, Calif.), or, in the alternative or in addition, selection for HLA-DR expression, Ghanekar et al., J. Immunol. 157:4028-4036 (1996).
These serial manipulations, however, may substantially alter the DC cell phenotype from that present in vivo. For example, linxe2x88x92HLA-DR+CD123+ dendritic cells in fresh preparations of tonsillar mononuclear cells express low levels of the T cell costimulatory molecules CD80 (B7.1), CD86 (B7.2), and HLA-DQ. Even an overnight culture of these cells in the absence of added cytokines is sufficient to induce the mature DC phenotype with upregulation of CD86, CD80, HLA-DQ and HLA-DR. Olweus et al., Proc. Natl. Acad. Sci. USA 94(23): 12551-12556 (1997). Longer term culture of CD34+ dendritic cell precursors in the presence of cytokines effects substantial phenotypic changes. Caux et al., J. Exp. Med. 184:695, 1996; Olweus et al., Proc. Natl. Acad. Sci. USA 94(23):12551-12556 (1997).
Thus, there exists a need in the art for methods of assaying dendritic cells without prior immunopurification or in vitro culture.
The paucity of DC-specific cell surface markers further suggests that surface immunophenotypic markers may only incompletely distinguish dendritic cell subsets that are, nonetheless, functionally distinct. For example, peripheral blood dendritic cells have been shown to fall into two subsets distinguishable by the divergent expression of CD11c and CD123: one subset is CD11c+CD123low, the other CD11cxe2x88x92CD123+. Olweus et al., Proc. Natl. Acad. Sci. USA 94(23): 12551-12556 (1997). Yet the critical and disparate roles that dendritic cells play in the immune system would argue that these two subsets each likely encompasses a variety of cell types with disparate functional activity.
There thus exists a need in the art for methods of distinguishing dendritic cell subsets using phenotypic criteria other than, or in addition to, expression of cell-surface markers. There further exists a need for methods of subsetting DC based on criteria that may be related more directly to DC function.
Recently, several groups have reported that intracellular staining of cells using cytokine-specific antibodies permits the flow cytometric analysis of cytokine expression in highly purified blood cell lineages, including purified dendritic cells. Picker et al., Blood 86(4):1408-1419 (1995); Waldrop et al., J. Clin. Invest. 99:1739-1750 (1997); Ghanekar et al., J. Immunol. 157:4028-4036 (1996); de Saint-Vis et al., J. Immunol. 160:1666-1676 (1998). More recently, Suni et al., J. Immunol. 212:89-98 (1998) described an assay for concurrent expression of intracellular cytokines and cell surface proteins in antigen-stimulated T lymphocytes without prior T cell purification. Similar assays are described and claimed in co-owned and copending U.S. patent application Ser. Nos. 08/760,447 and 08/803,702.
There exists a need in the art for a method that would adapt intracellular cytokine assays to the measurement of cytokine production by unpurified DC cells in whole blood.
The present invention solves these and other problems in the art by providing a flow cytometric method for measuring dendritic cell function in whole blood, comprising the steps of: (a) contacting a whole blood sample with a dendritic cell activator; (b) contacting the sample with a plurality of dendritic cell-distinguishing antibodies and at least one cytokine-specific antibody; and then (c) flow cytometrically assaying the sample for the binding of cytokine-specific antibody by at least one distinguishable DC subset.
In preferred embodiments, activation is performed in the presence of an inhibitor of protein secretion, and following permeabilization of the cells cytokines are detected intracellularly. Thus, in a particularly preferred embodiment, the dendritic cell activator contacting step is performed in the presence of brefeldin (gamma, 4-dihydroxy-2-(6-hydroxy-1-heptenyl)-4-cyclopentanecrotonic acid lambda-lactone) A, and the antibody contacting step itself comprises the steps, in order, of: (b1) adding a plurality of dendritic cell-distinguishing antibodies to the sample ; (b2) lysing erythrocytes in the sample; (b3) permeabilizing nucleated cells in the sample; and then (b4) adding at least one cytokine-specific antibody to the sample.
The dendritic cell-distinguishing antibodies may include a plurality of non-DC lineage-specific antibodies. In such cases, it is particularly preferred that each of the non-DC lineage-specific antibodies be conjugated to the identical fluorophore. When a plurality of non-DC lineage-specific antibodies is used, the dendritic cell-distinguishing antibodies further include an antibody specific for HLA-DR.
In a preferred embodiment, subsets of dendritic cells are distinguishably labeled. In this embodiment, the dendritic cell-distinguishing antibodies include at least one antibody that binds differentially to the surface of the different dendritic cell subsets. Particularly preferred in this embodiment is the use of antibody specific for CD11c or CD123.